Ca2????dependent protein kinase II (CaMKII) is the most abundant protein kinase in the mammalian brain and has been ascribed numerous functions including the regulation of neuronal process outgrowth, stabilization of synapse structure, and in the initiation and maintenance of short and long-term synaptic plasticity. CaMKII is a twelve-subunit complex that interacts with a variety of synaptic proteins in vitro. Its unique structure and multivalent nature have led to the hypothesis that, in addition to its role as a protein kinase, CaMKII plays a role as a scaffolding protein that helps assemble the necessary protein machinery that subserves synaptic function. This project focuses on CaMKII's role in the post-synaptic spine compartment and specifically, how it is involved in the assembly and regulation of the post-synaptic density (PSD) and the actin cytoskeleton. Four Aims are proposed. In Aim 1, we will use immunogold labeling and electron tomography to establish the spatial relationships (lateral and laminar) between CaMKII and a subset of signaling and interacting proteins. This will be the first analysis of the 3D disposition of many of these proteins within the PSD and we hypothesize that organized ensembles of signaling proteins exist that will be revealed in these studies. Aim 1 will further investigate CaMKII phosphorylation and dephosphorylation in the PSD and examine structural changes associated with these modifications. In Aim 2, we will take advantage of the fact that distinct CaMKII binding partners are expressed at different developmental stages and examine when and where individual proteins are added to the PSD. We will also determine where in the PSD these proteins are recruited in relation to CaMKII to assess how signaling modules might be assembled. In Aim 3 we will isolate PSDs from hippocampal slices where synapses have been activated. The impact of altered activity on the spatial distribution of CaMKII and other CaMKII interacting partners and signaling modules will be analyzed. Stimulation will also be applied in the presence or absence of CaMKII and phosphatase inhibitors to assess the role of acute activation of CaMKII on the structure and organization of proteins within the PSD. Changes in the structure of the PSD are likely organized with changes in spine architecture that are regulated by the actin cytoskeleton and CaMKII is a potential molecule that could coordinate these changes. In Aim 4, we will examine the interaction of CaMKII with actin and actin filaments and how the interaction is influenced by Ca2? binding and the autophosphorylated state of the enzyme. We will extend these analyses to all four mammalian isoforms of CaMKII. Lastly, we will produce tomographic reconstructions of CaMKII bound to actin filaments to establish whether CaMKII can both cross-link actin filaments as bundles and/or serve as a linker for orthogonal organization of actin filaments. Successful completion of the Specific Aims will provide a wealth of nanometer scale anatomical data concerning the organization of proteins within synaptic spines and add an in-depth understanding for CaMKII's role in regulating synaptic function. PUBLIC HEALTH RELEVANCE: Synapses are the site of chemical transmission in the central nervous system and proper formation and function of synaptic contacts is essential for brain function. Modification of these synaptic contacts underlies learning and memory and the lack of or excessive modification of synapses leads to problems such as epilepsy, Parkinson's disease, Huntington's disease, Alzheimer's disease and a variety of other catastrophic nervous system diseases. Specific molecules, like CaMKII, the focus of this proposal, are thought to coordinate and regulate the formation and modification of synaptic connections and our goals are to understand how this happens using a variety of modern techniques such as structural biology, protein chemistry and biophysics.